An alternative fast and convenient genotyping method for the screening of angiotensin converting enzyme gene polymorphisms.

Insertion/deletion (I/D) polymorphisms in intron 16 of the angiotensin converting enzyme gene (ACE) are associated with the plasma angiotensin converting enzyme (ACE) levels, and individuals with the DD allele have been reported to be more susceptible to cardiovascular disease than those without. The conventional genotyping method for the screening of I/D polymorphisms, which involves polymerase chain reaction (PCR)-gel electrophoresis, is laborious and time-consuming. In this study, we assessed the use of TaqMan-PCR genotyping for the screening of I/D polymorphisms as a replacement for the conventional method. We genotyped seven single nucleotide polymorphisms (SNPs) in linkage disequilibrium (LD) with the I/D polymorphisms, and calculated the LD coefficients of the I/D polymorphisms. We found that three polymorphisms, rs4331, rs4334 and rs4341, exhibited the highest LD coefficients (D' = 1.000; r2 = 0.967) and that the genotyping of rs4341 by the TaqMan-PCR method yielded the best discrimination among the different genotypes. Genotyping of 511 samples took only 2 h and the amount of DNA required for each test was only 6 ng by the TaqMan-PCR method using rs4341. In the course of this study, we identified a novel additional polymorphism (a deletion of six amino acids) in exon 13, near rs4316. The deletion allele encoded the testicular ACE, but not the plasma ACE. We concluded that genotyping of the rs4341 ACE polymorphism by the TaqMan-PCR method is a fast and convenient alternative method for direct I/D genotyping. We also concluded that testicular ACE may manifest a deletion of six amino acids that may result in deleterious function of this enzyme.     

Is there an association between angiotensin-converting enzyme gene variants and chronic nonproductive cough?

BACKGROUND: It is unclear why some patients develop a chronic nonproductive cough. Angiotensin-converting enzyme (ACE) inactivates tussive peptides in the airways such as bradykinin and tachykinins. An insertion/deletion polymorphism in the ACE gene accounts for variation in ACE levels, and patients with the II genotype have lowest serum ACE levels compared with ID and DD genotypes. We hypothesized that the II genotype would be associated with increased risk of developing a chronic cough. MATERIALS AND METHODS: We recruited 47 patients (33 women), referred for evaluation of cough (median cough duration, 24 months; range, 2 to 240 months). Cough patients were evaluated using a comprehensive diagnostic protocol, and cough reflex sensitivity was measured using a capsaicin inhalation challenge. ACE genotyping was performed on DNA samples from patients using the polymerase chain reaction followed by agarose gel electrophoresis. ACE genotypes in patients with chronic cough were compared with those in 199 healthy control subjects. Serum ACE levels were determined using a colorimetric assay. RESULTS: Genotype frequencies for the ACE gene were similar between patients and control subjects. There was no correlation between capsaicin sensitivity and ACE genotypes or serum ACE levels. CONCLUSION: Susceptibility to develop chronic cough is not associated with ACE genotype.     

血管紧张素转换酶基因多态性与阻塞性睡眠呼吸暂停综合征

目的 探讨血管紧张素转换酶（ＡＣＥ）基因多态性与阻塞性睡眠呼吸暂停综合征（ＯＳＡＳ）之间的关系。方法 以ＡＣＥ基因内含子１６的一个２８７ｂｐ的Ａｌｕ顺序Ｉ／Ｄ（ｉｎｓｅｒｔｉｏｎ／ｄｅｌｅｔｉｏｎ）型为我希望标志，用聚合酶链反应（ＰＣＲ）扩增基因片段，对５０例ＯＳＡＳ患和５０名正常人ＡＣＥ基因多态性的分布进行了观察，并结合了观察，并结合临床特点和多导睡眠监测结果进行了分析。结果 发现在中国汉族正     

Detection of angiotensin-converting enzyme gene insertion/deletion polymorphism from paraffin-embedded tissues: the Hisayama study.

Previous studies have suggested that archival materials from formalin-fixed paraffin-embedded blocks are unsuitable for most molecular techniques because the extracted DNA can be severely degraded. Therefore, the present study was designed to investigate the accuracy of genotyping for the insertion (I)/deletion (D) polymorphism of the angiotensin-converting enzyme (ACE) gene from paraffin-embedded tissues of autopsy cases from Hisayama Town, Japan. The genotype was determined using the double polymerase chain reaction method and to test the accuracy of the method, the polymorphism was investigated using paraffin-embedded tissues from 18 cases whose ACE genotypes (6 cases for each genotype) were known in advance from analysis of fresh-frozen tissue samples. Genotyping using paraffin-embedded tissues was then determined for 968 autopsy subjects. The genotype could be determined in 16 of the 18 test samples (88.9%) and there was no discrepancy with the results obtained from the fresh-frozen tissues. Of the 968 autopsy cases, the frequency of the DD, ID, and II genotypes was 12.4%, 47.3%, and 40.3%, respectively, a distribution that did not deviate from the Hardy-Weinberg equilibrium (chi(22df )= 0.67, p=0.72). These findings suggest the accuracy of the present method of ACE genotyping from paraffin-embedded tissues.     

糖尿病合并高血压脑卒中患者血管紧张素转换酶基因多态性的观察

目的研究血管紧张素转换酶（ＡＣＥ）基因的插入多态性与糖尿病合并高血压脑卒中发病的关系。方法应用ＰＣＲ扩增方法鉴定５０例糖尿病并高血压脑卒中患者、６９例糖尿病无血管病变患者和８５例健康对照者的ＡＣＥ基因多态性。结果糖尿病并高血压脑卒中患者ＡＣＥ基因的插入／插入纯合型频率（４６％）明显高于对照组（３０％）（＜Ｐ０．０５）；插入／缺失杂合型及缺失／缺失纯合型的频率（分别为２８％和４８％）与对照组（分别为２６％和２２％）差异无显著性（均为Ｐ＞０．０５）。糖尿病无血管并发症患者ＡＣＥ基因各型与对照组差异无显著性（均为Ｐ＞０．０５）。脑ＣＴ扫描显示基因插入／插入纯合型的糖尿病并高血压脑卒中患者中，７６％有多发腔隙性脑梗塞。结论ＡＣＥ基因插入／插入纯合型与脑卒中发病相关。     

血管紧张素转化酶抑制剂所致咳嗽与血管紧张素转化酶基因型相关性的研究

目的　探讨老年原发性高血压患者口服血管紧张素转换酶抑制剂 (ACEI )后发生咳嗽的机制。方法　应用聚合酶链反应 (PCR) ,检测老年原发性高血压患者口服ACEI后发生咳嗽与无咳嗽者的血管紧张素转化酶 (ACE)基因多态性 ,检测并比较两组患者血清ACE水平及ACE水平预测高血压患者口服ACEI引起咳嗽的敏感性和特异性。结果　ACEI所致咳嗽组ACE基因Ⅱ型的频率为4 0 % ,显著高于无咳嗽组 (2 0 % ,P <0 0 5 ) ,Ⅰ等位基因频率为 6 0 % ,显著高于无咳嗽组 (4 1% ,P <0 0 1)。两组患者血清ACE水平在DD型、ID型、Ⅱ型依次减低。咳嗽组血清ACE水平显著低于无咳嗽组 (P <0 0 0 1) ,血清ACE水平预测ACEI引起咳嗽的敏感性和特异性分别为 81%和 78%。结论　老年高血压患者口服ACEI所致咳嗽与血清ACE水平及ACE基因多态性有关。     

Angiotensin Converting Enzyme Gene Polymorphism Is Not Related to Essential Hypertension in a Greek Population

Studies in various ethnic groups have shown contradictory evidence on the association of the angiotensin converting enzyme (ACE) insertion/deletion (I/D) polymorphism with essential hypertension. In addition, mistyping of the insertion allele in heterozygotes has been reported. We analyzed the ACE genotype of 98 hypertensive and 84 normotensive subjects of Greek origin. Genomic DNA was extracted from blood samples and amplified by polymerase chain reaction (PCR). PCR primers were flanking the polymorphic region in intron 16 of the ACE gene. To avoid mistyping of heterozygotes, samples with the DD genotype were also amplified with primers that detect only the insertion allele. The distribution of the DD, ID, and II ACE genotypes was 30, 45, and 23 in hypertensive patients and 29, 40, and 15 in normotensive subjects, respectively. The estimated frequency of the insertion allele was 0.45 in hypertensive and 0.42 in normotensive subjects. The difference was not statistically significant. The results indicate a lack of association between ACE I/D polymorphism and essential hypertension in this Greek population, suggesting that other genes must contribute to the pathogenesis of hypertension. Am J Hypertens 1996; 9:700–702     

Angiotensin converting enzyme gene polymorphisms and coronary risk in a Portuguese population]

BACKGROUND: A family history of coronary heart disease (CHD) is a strong risk marker for the disease, independently of classical risk factors. It could be decoded by recognizing the polymorphisms associated with increased risk. Renin-angiotensin system genes are candidate genes in CHD and the deletion allele of the angiotensin converting enzyme (ACE) has been reported as deleterious. However, there is disagreement as to the role of the insertion/deletion polymorphism of the ACE gene in coronary risk. AIM: To evaluate whether ACE gene polymorphisms constitute a CHD risk factor. METHODS: We conducted a population-based case-control study of 301 subjects with a history of myocardial infarction or angiographic evidence of coronary heart disease and 510 age- and gender-matched controls, without CHD, living in a region with high CHD mortality rates. Blood samples were taken, DNA extracted and genotypes determined by the polymerase chain reaction (PCR). Amplification products were identified by agarose gel electrophoresis. STATISTICAL ANALYSIS: The Data were evaluated by SPSS for Windows, using the Student's t test, the chi-square test, odds ratios and 95% confidence intervals. RESULTS: The prevalence of the DD, ID and II genotype was 41.2%, 46.3%, 12.5% in the cases and 28.1%, 55.2% and 16.7% in the control group. The frequency of the DD genotype was significantly higher in the cases than in the controls (41.2% vs. 28.1%, odds ratio 1.79, 95% CI 1.31 to 2.4, p < 0.0001). By contrast, the ID and II genotypes' prevalence was higher in the control group (55.2% vs. 46.3%, p = 0.002 and 16.7 vs. 12.5%, p = NS, respectively) compared to the case group. CONCLUSIONS: This study clearly shows that the ACE DD polymorphism is strongly linked to CHD, and if our data are confirmed in a larger population sample, more aggressive vascular prevention could be justified in patients carrying the DD genotype.     

Rapid determination of the Delta32 deletion in the human CC-chemokine receptor 5 (CCR5) gene without DNA extraction by lightcycler real-time polymerase chain reaction

CCR5 is a major coreceptor for cellular entry of macrophage-tropic isolates of the human immunodeficiency virus (HIV). A 32-base pair deletion of the CCR5 gene (CCR5Delta32) protects against HIV infection because the frame shift leads to a truncated protein not expressed on the cell surface. CCR5Delta32 also delays progression in heterozygous HIV-infected patients and improves responses to antiretroviral therapy. Available methods for CCR5 genotyping, however, are cost expensive and/or time consuming. To improve CCR5 genotyping we studied four primer sets flanking the CCR5Delta32 deletion site using real-time polymerase chain reaction (PCR) on a LightCycler device. Primers amplified fragments of different length depending on the presence or absence of the Delta32 mutation. Next, melting curves of the amplified fragments were analyzed using SYBR green, a conventional double-strand DNA dye. To circumvent initial DNA extraction, we also studied serially diluted "interphase" leukocytes as PCR templates after centrifugation of EDTA blood. Finally, the validity of the new method was checked by analyzing 100 blood samples with known CCR5 genotypes. Amplicons of 82 bp:50 bp and 97 bp:65 bp fragment ratios could easily be discriminated due to the differences in their melting temperatures (3 degrees C and 2 degrees C, respectively). Furthermore, CCR5 genotyping was possible without initial DNA extraction and yielded optimal results at 1:400 to 1:600 dilution of the "interphase" leukocytes. Results of the new LightCycler PCR protocol were identical to conventional CCR5 genotyping but required considerably less time and expenditures. We have established a new real-time PCR protocol, which enables fast, cost-saving, and reliable CCR5 genotyping.     

Abnormalities of the p53 gene in juvenile myelomonocytic leukaemia

Juvenile chronic myelomonocytic leukaemia (JMML) is a rare myeloproliferative disorder of childhood. Fewer than 30% of cases of JMML terminate in a blast crisis; however, its molecular mechanism is unknown. Since mutation and/or deletion of the p53 gene has been reported to be associated with disease progression in a wide variety of human cancers, including adult-type chronic myelogenous leukaemia, we studied the p53 gene in 20 patients with JMML (16 samples in chronic phase and seven at blast crisis). Exons 4-8 of the p53 gene, which cover all the hot spots of point mutations, were amplified by the polymerase chain reaction (PCR) method and subjected to mutation screening by single-strand conformation polymorphism analysis. No mobility shift of single-strand DNA of PCR products in polyacrylamide gel electrophoresis, indicating point mutations, was found in 19/20 patients. DNA of the remaining patient in the chronic phase failed to be amplified by PCR and Southern blot analysis with XbaI-digested genomic DNA revealed a gross rearrangement (presumed deletion) of the p53 gene. These data indicate that abnormalities of the p53 gene are rare in JMML and not responsible for acute transformation, but could be involved in the pathogenesis of some cases of JMML.     

High frequency of DD polymorphism of the angiotensin-converting enzyme gene in Turkish asthmatic patients.

The aim of this study is to detect the incidence of the angiotensin-converting enzyme (ACE) gene polymorphism in Turkish asthmatic patients and to examine whether there is an association between the disease and ACE gene polymorphism. In our study, the genomic DNA of 100 asthmatic patients and 88 healthy subjects was analyzed Genomic DNA was isolated from peripheral blood by using standard methods. The intron 16 of the ACE gene was amplified by the polymerase chain reaction (PCR) method using primers ACE and ACEX to examine the presence and absence of a 287-base pair (bp) DNA fragment that showed I/D polymorphism genotypes. PCR products were separated by agarose gel electrophoresis and were visualized by a charge-coupled device camera. Serum ACE activities were measured using an ACE kit. The results were evaluated statistically using the chi-square test and one-way analysis of variance. Although the population of patients with asthma was characterized by a higher frequency (30%) of the DD genotype of ACE, they were characterized by lower frequency (48%) of the ID genotype of ACE (DD, 16%, and ID, 64%, in healthy control subjects). The frequency of the I and D alleles of the ACE gene was not significantly different between asthmatic patients (0.46/0.54) and healthy controls (0.52/ 0.48). In addition, in both asthmatic patients and controls, there was a significant decrease of the levels of ACE activity in individuals that have II genotypes when compared with individuals that have DD genotypes. ACE activities were increased significantly in all asthmatic patients (67.20 +/- 1.95 IU/L) compared with all healthy controls (60.90 +/- 2.12 IU/L).     

The involvement of the renin-angiotensin system gene polymorphisms in coronary heart disease

INTRODUCTION AND OBJECTIVES: Previous studies angiotensin-converting enzyme gene insertion/deletion polymorphism ACE (I/D), angiotensinogen gene polymorphism, and angiotensin II AT1 receptor polymorphism in relation to coronary heart disease controversial results. This study was designed to analyze the association between these gene polymorphisms and the first coronary event in individuals residing on Grand Canary Island, Spain. PATIENTS AND METHOD: Case-control study. Case subjects (n = 304) were recruited at the first coronary event; age-matched controls (n = 315) were randomly selected from the Grand Canary population. Participants were examined for the usual risk factors. Blood samples were obtained for biochemical analyses and DNA extraction. Genotyping was performed by PCR and restriction analysis. RESULTS: Neither ACE (I/D) nor AT1 receptor polymorphism was associated with coronary heart disease, whereas the frequency distribution of AGT M235T genotypes among patients and control subjects (TT: 29% and 19%; MT: 48% and 50%; MM: 22% and 31%, respectively) was statistically different (p = 0.003). Multiple logistic regression analysis identified the TT genotype of the angiotensinogen gene (OR = 1.9; 95% CI 1.1-3.4), diabetes (OR = 4.4; 95% CI 2.0-9.4) and hypertension (OR = 2.1; 95% CI 1.3-3.3) as risk factors predicting the coronary event. CONCLUSIONS: Our results provide no evidence of an association between ACE (I/D) or AT1 receptor polymorphism and coronary heart disease. However, homozygosity for the T allele of the angiotensinogen gene, diabetes and hypertension independently place individuals at higher risk of experiencing a coronary event on Grand Canary Island.     

Angiotensin-converting enzyme gene I/D polymorphism in malignant hypertension

BACKGROUND: The mechanism of the rapid transition of a stable benign hypertensive disease to a severe and devastating malignant hypertension is not fully understood. However, the renin angiotensin system, which is highly activated in malignant hypertension, is established as an important pathogenetic factor in different cardiovascular and renal diseases. Over the last decade, a polymorphism in genes regulating this system has been found. This includes the 287 bp sequence deletion (D)/insertion (I) polymorphism in the angiotensin-converting enzyme (ACE) gene and the methionine (M) to threonine (T) point mutation polymorphism in the angiotensinogen (AGT) gene. These gene polymorphisms have been associated with various cardiovascular and renal diseases and the aim of this study was to investigate whether they were linked to malignant hypertension. METHODS: Forty-two patients with malignant hypertension (mean age 55 years), 42 patients with non-malignant hypertension (mean age 57 years) and 85 normotensive control subjects (mean age 42 years) were investigated with respect to ACE I/D and AGT M/T genotypes. DNA was prepared by standard methods from isolated white blood cells and analysed by the PCR technique. The PCR reaction used in the detection of the ACE I/D polymorphism was optimized for an equal amplification of the I and D alleles. RESULTS: The frequency of the DD genotype was significantly increased in patients with malignant hypertension (43%) compared with patients with non-malignant hypertension (14%) and normotensive control subjects (18%) (p <0.01) for both. The frequency distribution of AGT M/T genotype did not differ between patients with malignant and non-malignant hypertension. CONCLUSION: The DD genotype of the ACE gene occurred more than twice as often in malignant hypertension than in non-malignant hypertension and indicates that ACE gene polymorphism is a significant risk factor for initiation of malignant hypertension.     

Angiotensin-converting enzyme gene polymorphism in relation to HLA-DR in sarcoidosis

OBJECTIVES: To investigate if an insertion/deletion (I/D) polymorphism in the angiotensin-converting enzyme (ACE) gene associates with HLA-DR alleles previously found to be of prognostic interest in Scandinavian sarcoidosis patients. This may contribute to characteristics associated with these HLA-DR alleles, such as a good (DR17) or poor (DR14 or 15) prognosis. DESIGN, SETTINGS AND SUBJECTS: Polymerase chain reaction (PCR) was used for analysing an I/D polymorphism in the gene coding for ACE in 138 subjects; 65 controls and 73 sarcoidosis patients, and for HLA-DR genotyping 67 patients. Serum ACE level (S-ACE) was measured in all controls and 72 patients. Sixty-one patients were classified as chronic or nonchronic after 2 years follow-up. All patients were recruited and followed at our outpatient clinic. RESULTS: No significant differences in ACE alleles or genotypes were seen between controls and patients or between patients positive and negative for DR17 or DR14/15. The ACE genotype did not differ between nonchronic and chronic patients. The ACE genotype tended to influence the S-ACE in patients, whilst in controls S-ACE significantly differed between the ACE genotypes. CONCLUSION: This study does not support an association between ACE genotype and sarcoidosis or disease outcome. However, because significantly (P < 0.001) more DR17 positive (17 of 19) than DR14/15 positive (seven of 26) patients were classified as nonchronic, these results instead strengthen the prognostic importance of HLA-DR alleles in Scandinavian sarcoidosis patients.     

Association of low density lipoprotein receptor related protein-associated protein (LRPAP1) gene insertion/deletion polymorphism with gallstone disease

BACKGROUND AND AIM: Gallstones are byproducts of cholesterol supersaturated bile. Various studies have indicated that there might be a genetic predisposition to the disease. Receptor-associated protein (RAP) is a molecular chaperone for low density lipoprotein receptor-related protein (LRP), which plays a key role in cholesterol metabolism. Intron 5 insertion/deletion polymorphism of RAP gene (LRPAP1) has been implicated in other diseases sharing etiology with gallstone disease (GSD). METHODS: To analyze the association of insertion/deletion polymorphism in GSD, 130 gallstone patients and 202 healthy subjects took part in the present study. For genotyping, polymerase chain reaction was followed by 2% agarose gel electrophoresis. RESULTS: The results showed that frequencies of D and I allele were 65.77% and 34.23% in patients, 76.24% and 23.76% in controls, respectively. Frequency of I allele was significantly higher in the patient group than in the control group (P = 0.003). CONCLUSION: In the present study I (insertion) allele was found to be associated with GSD.     

Linkage analysis of angiotensin-converting enzyme (ACE) insertion/deletion polymorphism and systemic lupus erythematosus

Previous studies have suggested an association between systemic lupus erythematosus (SLE) and an insertion/deletion polymorphism in the angiotensin-converting enzyme gene (ACE). This polymorphism consists of a 250-bp insertion/deletion of an alu repeat in the 16th intron of the ACE gene. Individuals homozygous for the deletion have a higher level of circulating enzyme. Due to the important role of this enzyme in regulating the renin--angiotensin and kallikrein--kininogen systems, it is possible that the ACE insertion/deletion may play a role in SLE, which can include vasculitis and vascular changes. Using primers flanking the insertion/deletion site, we have examined the ACE gene in lupus patients and family members using genomic DNA obtained from the Lupus Multiplex Registry and Repository (LMRR). We were unable to detect significant linkage or genetic association between the ACE gene and SLE.     

老年脑卒中患者血浆ACE水平及其基因多态性的研究

血管紧张素转换酶(ACE)在肾素-血管紧张素-醛固酮系统(RASS)和激肽释放内原-激肽系统(KKS)中发挥着重要的作用。近年研究发现,ACE通过不同的遗传机制在心脑血管疾病的发生发展中发挥作用,其多态性决定了血浆和细胞内ACE浓度,是研究各类心脑血管疾病遗传易感性的候选基因,本文通     

微小隐孢子虫卵囊DNA提取及用于PCR检测

目的采用3种方法提取微小隐孢子虫卵囊DNA,并用于PCR检测以进行比较。方法 微小隐孢子虫卵囊经多次冻融加热破壁后,采用螯合树脂(Chelex-100)、酚/氯仿和基因组DNA纯化系统试剂盒3种方法提取微小隐孢子虫卵囊DNA,并根据微小隐孢子虫基因序列(L16996)设计一对寡核苷酸引物,分别对3种方法制备模板进行PCR扩增分析。Chelex-100提取的DNA也用于观察PCR检测的敏感性。结果3种方法制备的微小隐孢子虫卵囊模板用于PCR检测均获得1条446 bp条带,Chelex-100提取的DNA用于PCR检测的敏感性至少达0.5个卵囊。结论3种方法提取的微小隐孢子虫卵囊DNA均可用于PCR检测,Chelex-100法是一种高效而快速的微量提取DNA方法,适用于对隐孢子虫DNA的检测。     

乙型肝炎病毒前S2序列异质性的初步研究

目的 通过对乙型肝炎病毒（HBV）前S2基因序列的研究,证实HBV在慢性感染者中以准种状态存在,并对前S2序列异质性进行初步的阐明。方法 以中国株HBV基因序列为依据,设计特异性PCR引物,自51例慢性HBV感染患者血清中扩增HBV前S2基因序列,采用聚丙烯酰胺凝胶电泳技术展示缺失突变,随机选择克隆测序,确定病毒的变异程度。结果 聚丙烯酰胺凝胶电泳结果发现,52.9%（27／51）患者血清中可获得2或3条扩增条带;测序结果发现前S2基因序列存在缺失突变高发区,氨基酸序列分析示缺失突变主要位于前S2编码蛋白的氨基端。结论 HBV前S2序列内有一缺失高变区,并在患者体内表现为多种变异形式;氨基端附近的变异可能影响中和抗体的识别作用。结果提示HBV慢性携带者体内有HBV准种共存。